Reg-like protein

ABSTRACT

Methods of detecting the presence of a tumor or a cancerous condition includes detecting the expression of RELP proteins, related polypeptides and proteins, or nucleic acid molecules indicative of such expression are presented. These methods include antibody-based assays and molecular diagnostic assays such as PCR-based methods.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. 119(e) ofprior application Ser. No. 60/276,414, filed Mar. 16, 2001, nowabandoned.

BACKGROUND

The invention relates to proteins whose presence, absence, concentrationin biological samples, or expression level can be used to indicate thepresence or absence of tumors, cancer, and related conditions as well asrelated prognoses and diagnoses.

Tumors are abnormal masses of tissue. When tumors proliferateuncontrollably, they are said to become malignant. This condition isgenerally referred to as a cancer. Numerous methods are used todetermine when a patient has developed a tumor and when the conditionhas become cancerous. The identification or quantitation of varioustumor or cancer markers is one desirable means for making suchdeterminations.

Broadly, a “marker” is any property that can be used to distinguishcancer from normal tissues and from other disease states. The markers'presence is then a basis for classification. More specifically, the termis used to denote particular molecules that are amenable to assay. Serummarkers, as the name implies, are markers that are readily assayed inthe serum of a patient. Typically, they are secreted proteins or cellreceptors that are abundant in tumor cells well beyond their presence(or total absence) in normal cells and tissues. Examples include PSA,CEA, and AFP.

A more expansive consideration of tumor and cancer markers includes thedetection of tumors and cancer from the nucleic acids produced invarious cells (as well as other materials that are related to nucleicacids). Cancer is generally considered to be a disease of multiplemutations. Thus, detection of the mutations at the molecular leveloffers the prospect of more direct and more reliable diagnoses than waspossible with some of the older cancer markers. Thus, it is appropriateto consider a nucleic acid sequence that is indicative of the mutationthat causes or occurs with the cancerous condition to be a cancermarker. The ability to conduct nucleic acid analyses does not vitiatethe value of serum markers, however. Each may have an appropriate roleto play in the diagnosis, staging, and treatment monitoring of apatient.

Discovering genes that encode cancer-associated antigens and events alsoopens the door to genetic intervention against cancer cellproliferation. The accurate and consistent use of a cancer marker todifferentiate cancerous from normal tissue, not only has diagnosticpotential, but is also desirable for treatment and prognosis. Therefore,such markers continue to be sought.

The reg proteins, which belong to the C-type lectin superfamily, aresecreted proteins of about 20 kD in size. They are found in normal andmalignant tissues of the gastrointestinal tract, in the pituitary and inregenerating neurons. Reg expression associates with cell proliferation,migration and differentiation (Chiba T et al., 2000, J Gastroenterol 35Suppl 12:52, Levine J L, 2000, Surg Res 89:60, Otonkoski T et al., 1994,Diabtets 43:1164, Bernard-Perronese FR, 1999, J Histochem Cytochem47:863). The known reg genes cluster on human chromosome 2p12. The firstcharacterized member of the reg protein family was Reg 1α, which wasisolated from rat regenerating pancreatic islets (Terazono et al.,1988). Subsequently, cDNAs encoding for four additional human regproteins, and the corresponding mouse and rat orthologs, have beencloned (Watanabe et al., 1990; Lasserre et al., 1992; Bartoli et al.,1993; Rafaeloff et al., 1997). They exert mitogenic activity to subsetsof epithelial and neuroectodermal cells (Katsumata et al., 1995,Zenilman et al., 1996; 1997; 1998; Livesey et al., 1997). A growthsignal transducing receptor for rat reg1 proteins was recentlydescribed. The receptor is encoded by a gene homologous to humanmultiple exostoses gene. It was found to have been expressed, inaddition to pancreatic islets, in various tissues including kidney,liver, gut, the adrenal and pituitary glands (Kobayashi S et al. 2000).

Identification, isolation, and use of new tumor and cancer markersremain important in the diagnosis, treatment and prevention of cancer.

BRIEF SUMMARY OF THE INVENTION

The invention is an isolated nucleic acid molecule that encodes RELPprotein. The molecule can be a nucleic acid molecule of Seq ID No 1, anucleic acid molecule encoding a protein having at least a 70% identityto a polypeptide comprising amino acids of SEQ ID NO:2.

The invention also encompasses a nucleic acid molecule that iscomplementary to the molecule that encodes a protein having at least 70%identity to Seq. ID No. 2, a nucleic acid molecule of at least 15sequential bases of the nucleic acid sequence of Seq. ID No. 1, or anucleic acid molecule that hybridizes under stringent conditions to thenucleic acid sequence molecule of Seq. ID No. 1.

In another aspect of the invention, isolated RELP is presented.

In yet another aspect of the invention methods of detecting the presenceof a tumor or a cancerous condition includes detecting the expression ofpolypeptides, proteins, or nucleic acid molecules having the sequencesdescribed above and correlating the presence or concentration of suchmolecule in a biological sample with the presence or absence of saidtumor or cancerous event.

In yet another aspsect of the invention, antibodies that binds to theRELP and functional equivalents thereof are presented.

In yet another aspect of the invention, kits for detecting thepolypeptides, proteins, or nucleic acid sequences described above arepresented.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleic acid sequence of the cDNA that encodes for RELP(Seq. ID No. 1).

FIG. 2 is the amino acid sequence of RELP (Seq. ID No. 2).

FIG. 3 is the nucleic acid sequence of the cDNA that encodes for RELPsignal protein (Seq. ID No. 3).

FIG. 3 a is the amino acid sequence of RELP signal protein (Seq. ID No.4).

FIG. 4 is a scaled schematic representation of the RELP gene.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

The term “protein superfamily” as used herein refers to proteins whoseevolutionary relationship may not be entirely established or may bedistant by accepted phylogenetic standards, but show similar threedimensional structure or display unique consensus of critical aminoacids. The term “protein family” as used herein refers to proteins whoseevolutionary relationship has been established by accepted phylogenicstandards.

As used herein, the term nucleic acid sequence includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“nucleic acid sequences” as that term is intended herein. Moreover, DNAsor RNAs comprising unusual bases, such as inosine, or modified bases,such as tritylated bases, to name just two examples, are nucleic acidsequences as the term is used herein. It will be appreciated that agreat variety of modifications have been made to DNA and RNA that servemany useful purposes known to those of skill in the art. The termnucleic acid sequence as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of nucleic acid sequences,as well as the chemical forms of DNA and RNA characteristic of virusesand cells, including simple and complex cells, inter alia. Nucleic acidsequences embraces short nucleic acid sequences often referred to asoligonucleotide(s).

As used herein, a “functional derivative” of RELP is a compound thatpossesses a biological activity (either functional or structural) thatis substantially similar to the biological activity of RELP. The term“functional derivatives” is intended to include the “fragments,”“variants,” “degenerate variants,” “analogs” and “homologues” or to“chemical derivatives” of RELP. A molecule is “substantially similar” toRELP if both molecules have substantially similar structures or if bothmolecules possess similar biological activity.

A newly identified protein, “RELP” (Reg Like Protein), is characterizedin this specification. Nucleic acids (including, for example, cDNA)encoding for this protein have been isolated and cloned and uses forthis protein in cancer diagnostics are presented. The gene structure andits chromosomal location are presented, and the tissue distribution ofits expression is described. Additionally, antibodies that bind to thisprotein have been prepared and methods for their use have been devised.The murine homologue of RELP was also cloned and characterized.

All nucleic acid sequences described in this specification are shown inthe 5′→3′ direction unless otherwise indicated.

FIG. 1 shows the nucleic acid sequence of a cDNA (Seq. ID No. 1) used toproduce RELP. The RELP cDNA encodes a 158-amino acid protein with aputative 22-amino acid signal peptide (FIG. 3). The molecular weight ofRELP is about 18 kd, and the isoelectric point was calculated as 9.128.The aminoterminus of RELP is highly hydrophobic and contains a cleavablesignal sequence of 22 aminoacids. Human Reg proteins are 51–87%identical and 55–87% similar to each other, whereas RELP is 32–37%identical and 42–47% similar to them.

The primary structure of RELP is similar to that of the subgroup ofC-type lectin superfamily of proteins, which contain a singlecarbohydrate-recognition (CRD) domain. The CRD-associated four conservedand two optional cysteines involved in intramolecular disulphide bondsare all conserved in RELP. Residues 50–53 represent a putativeN-glycosylation site. The secondary structure of RELP is similar to thatof human Reg1α and the global folds of these proteins appear to berelated. The amino acid sequence of RELP is shown in FIG. 2 (Seq. ID No.2).

The RELP gene resides on chromosome 1 band p12–13.1 and spans about17,500 base pairs. It is comprised of seven exons. FIG. 4 shows aschematic representation of the gene with the distance between exonsscaled. The location of each exon is shown in Roman numerals.

Expression of RELP in normal tissues: RELP message is highly expressedin a subset of epithelial cells in the small intestine. This subset ofcells represents the intestinal neuroendocrine cells (verified bycolocalization of chromogranin). RELP mRNA is also seen in the stomach,various parts of the colon, where it is localized in the epithelialcells in the crypt bottom, the pancreas, the prostate and the testis.

Expression of RELP in diseased tissues: RELP is ectopically abundantlyexpressed in mucinous tumors originating from various organs, such asovary, stomach, colon, breast and pancreas. The expression of RELP mRNAappears to be extremely high in mucinous ovarian tumors. On a proteinlevel a high, uniform expression is seen in the epithelial cells frommucinous ovarian, stomach, colon and breast tumors. Intraductal mucinouspancreatic tumors also express RELP. These tumors are emerging as anewly identified entity of pancreatic disorders that predisposerecurrent pancreatitis. They are probably apt to become malignant.

Biological samples from a subject are used to determine whether cancercells are present in the subject. Examples of suitable samples includeblood and biopsy material. One method of diagnosis is to expose RNA fromcells in the sample to a labeled probe that is capable of hybridizing tothe RELP gene transcript, or a fragment thereof, under stringentconditions. Of course, the hybridizing conditions are altered to achieveoptimum sensitivity and specificity depending on the nature of thebiological sample, type of cancer, method of probe preparation, andmethod of tissue preparation.

After contacting the sample with the probe, the next step is determiningwhether the probe has hybridized with nucleotide sequences of the mRNAfrom the sample, from which the expression of the RELP gene is inferred,the presence at elevated levels being diagnostic of cancer.

Another diagnostic method is to contact a sample with antibodiesdirected to antigenic (i.e. RELP) peptides. These antibodies are usefulin the development of very specific assays for the detection of RELPantigen, and allow the tests to be carried out in many differentformats. Preferably, the antibodies are labeled monoclonal antibodies.Since RELP is a secreted molecule, detecting RELP antigen in bodyfluids, such as serum, plasma, cyst fluids, pancreatic juice, and urinecan be used to detect or follow-up RELP-expressing cancers. Typically,the protein is expressed between 100 and 1000 times in diseased tissues(as described above) compared with its normal expression levels.Accordingly, serum levels of 200 to 1000% those of normal levels will bedetected in the serum assays of this invention. Most typically, a serumlevel of about 250% that of normal RELP levels can be expected inpatients with colon cancer. Likewise, in molecular diagnostic tests inwhich mRNA expression levels are assayed, expression levels that are 150to 1000% those of normal levels indicate disease.

Purified biologically active RELP may have several different physicalforms. RELP may exist as a full-length nascent or unprocessedpolypeptide, or as partially processed polypeptides or combinations ofprocessed polypeptides. The full-length nascent RELP polypeptide may bepostranslationally modified by specific proteolytic cleavage events thatresults in the formation of fragments of the full length nascentpolypeptide. A fragment, or physical association of fragments may havethe full biological activity associated with RELP however, the degree ofRELP activity may vary between individual RELP fragments and physicallyassociated RELP polypeptide fragments.

Since there is a substantial amount of redundancy in the various codonsthat code for specific amino acids, this invention is also directed tothose DNA sequences that contain alternative codons that code for theeventual translation of the identical amino acid. For purposes of thisspecification, a sequence bearing one or more replaced codons will bedefined as a degenerate variation. Also included within the scope ofthis invention are mutations either in the DNA sequence or thetranslated protein, which do not substantially alter the ultimatephysical properties of the expressed protein. For example, substitutionof aliphatic amino acids alanine, valine, leucine and isoleucine;interchange of the hydroxyl residues serine and threonine, exchange ofthe acidic residues aspartic acid and glutamic acid, substitutionbetween the amide residues asparagine and glutamine, exchange of thebasic residues lysine and arginine and among the aromatic residuesphenylalanine, tyrosine may not cause a change in functionality of thepolypeptide. Such substitutions are well known and are described, forinstance in Molecular Biology of the Gene, 4^(th) Ed. Bengamin CummingsPub. Co. by Watson et al.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude, but are not limited to site directed mutagenesis, chimericsubstitution, and gene fusions. Site-directed mutagenesis is used tochange one or more DNA residues that may result in a silent mutation, aconservative mutation, or a nonconservative mutation. Chimeric genes areprepared by swapping domains of similar or different genes to replacesimilar domains in the RELP gene. Similarly, fusion genes may beprepared that add domains to the RELP gene, such as an affinity tag tofacilitate identification and isolation of the gene. Fusion genes may beprepared to replace regions of the RELP gene, for example to create asoluble version of the protein by removing a transmembrane domain oradding a targeting sequence to redirect the normal transport of theprotein, or adding new post-translational modification sequences to theRELP gene. Examples of altered properties include but are not limited tochanges in the affinity of an enzyme for a substrate or a receptor for aligand. All such changes of the nucleic acid sequence or polypeptidesequences are anticipated as useful variants of the present invention solong as they retain their functionality consistent with the original useof the nucleic acid sequence or polypeptide sequence of the presentinvention as described herein.

Identity or similarity, as known in the art, are relationships betweentwo or more polypeptide sequences or two or more nucleic acid sequences,as determined by comparing the sequences. In the art, identity alsomeans the degree of sequence relatedness between polypeptide or nucleicacid sequence sequences, as the case may be, as determined by the matchbetween strings of such sequences. Both identity and similarity can bereadily calculated (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). While there exist a number of methods to measureidentity and similarity between two nucleic acid sequences or twopolypeptide sequences, both terms are well known to skilled artisans(Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press,1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991; and Carillo, H., and Lipman, D., (1988)SIAM J. Applied Math., 48, 1073. Methods commonly employed to determineidentity or similarity between sequences include, but are not limited tothose disclosed in Carillo, H., and Lipman, D., (1988) SIAM J. AppliedMath., 48, 1073. Preferred methods to determine identity are designed togive the largest match between the sequences tested. Methods todetermine identity and similarity are codified in computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, GCG programpackage (Devereux, J., et al., (1984) Nucleic Acids Research 12(1),387), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., (1990) J. Molec.Biol. 215, 403).

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Manyamino acids, including the terminal amino acids, may be modified in agiven polypeptide, either by natural processes, such as processing andother post-translational modifications, but also by chemicalmodification techniques which are well known to the art. Even the commonmodifications that occur naturally in polypeptides are too numerous tolist exhaustively here, but they are well described in basic texts andin more detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Amongthe known modifications which may be present in polypeptides of thepresent are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Such modificationsare well known to those of skill and have been described in great detailin the scientific literature. c.f. PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993).

Included within the scope of the invention are nucleic acid sequencesthat are at least 70% identical over their entire length to a nucleicacid sequence encoding the polypeptide having the amino acid sequencesset out herein, and nucleic acid sequences which are complementary tosuch nucleic acid sequences. Alternatively, highly preferred are nucleicacid sequences that comprise a region that is at least 80% identical,more highly preferred are nucleic acid sequences at comprise a regionthat is at least 90% identical, and among these preferred nucleic acidsequences, those with at least 95% are especially preferred.Furthermore, those with at least 97% identity are highly preferred amongthose with at least 95%, and among these those with at least 98% and atleast 99% are particularly highly preferred, with at least 99% being themost preferred. The nucleic acid sequences which hybridize to thehereinabove described nucleic acid sequences in a preferred embodimentencode polypeptides which retain substantially the same biologicalfunction or activity as the polypeptide characterized by the RELP aminoacid sequences set forth herein. Preferred embodiments in this respect,moreover, are nucleic acid sequences that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Seq. Id No. 1. The presentinvention further relates to nucleic acid sequences that hybridize tothe herein above-described sequences. In this regard, the presentinvention especially relates to nucleic acid sequences that hybridizeunder stringent conditions to the herein above-described nucleic acidsequences. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences.

Nucleic acid sequences of the invention may be used as a hybridizationprobe for RNA, cDNA and genomic DNA to isolate full-length cDNAs andgenomic clones encoding the sequences of RELP set forth herein and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to them. Such probes generally will comprise at least 15bases. Preferably, such probes will have at least 30 bases and may haveat least 50 bases. Particularly preferred probes will have at least 30bases and will have 50 bases or less. For example, the coding region ofthe gene of the invention may be isolated by screening using the knownDNA sequence to synthesize an oligonucleotide probe. A labeledoligonucleotide having a sequence complementary to that of a gene of thepresent invention is then used to screen a library of cDNA, genomic DNAor mRNA to determine to which members of the library the probehybridizes.

The polypeptides of the present invention include the polypeptide ofSeq. ID No. 2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% identity to the polypeptide of Seq.ID No. 2, preferably at least 80% identity to the polypeptide of Seq. IDNo. 2, and more preferably at least 90% similarity (more preferably atleast 90% identity) to the polypeptide of Seq. ID No. 2 and still morepreferably at least 95% similarity (still more preferably at least 97%identity) to the polypeptide of Seq. ID No. 2 and also include portionsof such polypeptides with such portion of the polypeptide generallycontaining at least 30 amino acids and more preferably at least 50 aminoacids. Representative examples of polypeptide fragments of theinvention, include, for example, truncation polypeptides of Seq. ID No.2 or of variants or derivatives thereof, except for deletion of acontinuous series of residues (that is, a continuous region, part orportion) that includes the amino terminus, or a continuous series ofresidues that includes the carboxyl terminus or, as in double truncationmutants, deletion of two continuous series of residues, one includingthe amino terminus and one including the carboxyl terminus. Alsopreferred in this aspect of the invention are fragments characterized bystructural or functional attributes of the polypeptide characterized bythe sequences of Seq. ID No. 2.

EXAMPLES Example 1 Cloning and Sequencing of cDNAs

An EST that was abundantly expressed in mucinous ovarian tumor-derivedlibraries was identified. A full length cDNA insert, encoding for thepredicted preprotein based on the EST was acquired, cloned into thepSport vector, and verified by sequencing. Blasting with the human RELPcDNA in the NCBI EST data base yielded three highly homologous mousesequences. The corresponding clones (IMAGE clone IDs 717371, 1079498 and1096767) were acquired and sequenced. The putative mouse orthologue forRELP was cloned into the XbaI site in the pGEMA1bSVPA vector. MurineRELP and the nucleic acid that codes for it was found to have 66% aminoacid and 70% nucleotide sequence identity respectively to human RELP andits associated nucleic acid. Murine RELP was also found to have 43–45%similarity and 32%–37% identity to other mouse Reg sequences. Thenucleic acid sequence of cDNA used to express it is shown in FIG. 1(Seq. ID No. 1).

Example 2 Antibodies

A C-terminal RELP-derived peptide was synthesized (CAEMSSNNNFLTWSSNESeq. ID No. 5), coupled to keyhole limpet hemocyanin, and used toimmunize rabbits for production of polyclonal antibodies. The sera weretested for reactivity against the corresponding peptide with ELISA, andthe positive batches were affinity-purified. The purified antibodyspecifically detected the protein that has the peptide epitope in tissuesections. This was verified by complete abolishment of the signal if thecorresponding peptide is added simultaneously with the antibody. Inaddition to this polyclonal antibody, which works well inimmunohistochemistry, monoclonal antibodies able to detect the proteinin its natural fold were produced. To produce monoclonal antibodies, apurified antigen, produced in mammalian cells to ensure natural fold andposttranslational modifications, was generated. The antigen, RELP-IgGconstant part fusion protein, was expressed in mouse myeloma cells, andthe secreted protein was purified using the Fc part as bait. Thispurified antigen was recognized in Western blot by the C-terminalpolyclonal antibody, and by five other anti-RELP peptide antibodies(Below, Seq. ID No.6-Seq. ID No.11). The antigen was used to generatemouse monoclonal antibodies against RELP by selecting out of thepositive clones those that produced antibodies that reacted against RELPinstead of the IgG constant part.

Kits for the clinical identification of RELP can be readily fashionedemploying these and similar antibodies. Such kits would includeantibodies directed to RELP identification, appropriate indicatorreagents (e.g., enzymes, labels, and the like), and (optionally) otherreagents useful in the clinical application of such a kit such asdilution buffers, stabilizers, and other materials typically used insuch assays. The kits would be used to detect RELP in body fluids toscreen or follow-up RELP expressing cancers, and to screen the presenceof RELP protein in tissue samples.

CYGYFRKLRNWSDAELECQSYGNGA Seq.ID No.6 WIDGAMYLYRSWSGKSMGGNKHC Seq.IDNo.7 CAEMSSNNNFLTWSSNE Seq.ID No.8 CAEMSSNMNFLTWSSNECNKRQHFLCKYR Seq.IDNo.9 CEYISGYQRSQPIWIGLHDPQKRQQWQ Seq.ID No.10 CQSYGNGAHLASILSLKEASTIASeq.ID No.11

Example 3 Double Immunofluorescence Staining

Tissue sections of normal duodenal mucosa were double stained with thepolyclonal peptide antibody against RELP (1:30; 25 μg/ml) and amonoclonal antibody against chromogranin A (1:5000; 0.2 μg/ml Chemicon,Temecula, Calif.) followed by tetramethylrhodamineisothiocyanate-conjugated swine anti-rabbit immunoglobulins (DAKO) andfluorescein isothiocyanate (FITC)-conjugated goat anti-mouseImmunoglobulins (ICN/Cappel). For control stainings primary antibodieswere replaced with the IgG fractions of normal rabbit and mouse sera.

The colocalization of RELP and chromogranin A indicates that theRELP-expressing cells in the duodenum belong to the neuroendocrinepopulation.

Example 4 In situ Hybridization

Formalin fixed paraffin embedded tissue samples were cut into 5–7 mμthick sections, mounted on silane coated glass slides, and incubated at37° C. over night and at 65° C. for 30 min before deparaffinating twicefor 10 min in xylene. Thereafter the samples were rehydrated through agraded series of ethanol solutions (100 to 70%), and rinsed twice for 5min in phosphate buffered saline (PBS pH 7.0), treated twice for 5 minwith 0.1 mol/L glysine in PBS, permeabilized for 15 min with 0.3% TritonX-100 in PBS. The sections were treated with proteinase K (Finnzymes,Helsinki, Finland) treatment (μg/ml, in TE buffer; 100 mmol/L Tris-HCl,50 mmol/L EDTA, pH 8.0) at 37° C. for 30 min, postfixed in 3%paraformaldehyde in PBS at 4° C. for 5 min and rinsed twice in PBS.Positive charges were blocked by soaking the slides in 0.25% (v/v)acetic anhydride, 100 mmol/L triethanolamine, pH 8.0, twice for 5 min.The slides were equilibrated in 4×SSC, 50% (v/v) deionized formamide at37° C. for 10 min. Probes were prepared by ligating a PCR-amplified 0.4kb RELP cDNA insert into the pCR-II vector using a TA cloning kit(Invitrogen, San Diego Calif., USA). The templates for RELP antisense orsense RNA probes were generated by linearizing the appropriate vectorconstruct (in 3′ to 5′ direction or 5′ to 3′ direction, respectively).An RNA Labeling Kit (Boehringer-Mannheim) was used to generatedigoxygenin labeled RNA probes by in vitro transcription. Thehybridization was performed overnight at 45° C. using a hybridizationmixture containing 1× Denhart's solution (0.2 g/L Ficoll Type 400,Pharmacia), 0.2 g/L polyvinylpyrrolidone, 0.2 g/L bovine serum albumin(fraction V; Sigma), 40% formamide, 10% dextran sulfate, 4×SSC, 10mmol/L dithiothreitol, 1 mg/mL yeast tRNA, 1 mg/mL herring sperm DNA and300 ng/mL digoxygenin-labeled RNA probe. After hybridization, the tissuesections were washed at 37° C. twice for 5 min in 2×SSC and once for 15min in 60% formamide, 0.2×SSC, followed by two 5 minute rinses in 2×SSCat room temperature and two 10 minute washes in 100 mmol/L Tris-HCl, pH8.0, 150 mmol/L NaCl. The signal detection was carried out using 1:250alkaline phosphatase-conjugated sheep antidigoxygenin fab fragments(Boehringer Mannheim). The signal was visualized by incubating thesections with NBT/BCIP Stock Solution (Boehringer Mannheim) for 1.5hours.

Small numbers of RELP-positive cells were seen in the gastric mucosa andin exocrine pancreas. In normal colon, RELP was localized in epithelialcells in the bottom of the crypts. A strong RELP mRNA signal was seen inthe cytoplasm of selected cells in the duodenal mucosa while most of theepithelium was negative. In mucinous cancers from ovary, stomach, colonand breast the RELP mRNA was also detected in the epithelial cells. Thevisualization of the RELP-specific mRNA confirmed that the RELP proteinwas expressed by these cells.

Example 5 Immunohistochemistry

An affinity-purified polyclonal antibody against the C-terminal peptideof RELP was used for the immunohistochemical detection and localizationof RELP. Four μm sections from formalin-fixed and paraffin embeddednormal and tumor tissue, obtained from the archives of the Department ofPathology, University of Helsinki, were mounted on3-aminopropyl-triethoxy-silane (APES, Sigma, St. Louis, Mo., U.S.A)coated slides. The sections were deparaffinized and rehydrated in gradedconcentrations of ethanol and treated with methanolic peroxide (0.5%hydrogen peroxide in absolute methanol) for 30 minutes at roomtemperature to block the endogenous peroxidase activity. Antigenretrieval was done in a microwave oven twice for 5 minutes (650W). AnElite ABC Kit (Vectastain, Vector Laboratories, Burlingame, Calif.,U.S.A) was used for immunoperoxidase staining. The RELP antibody wasused at an optimal dilution of 1:2000. Both the biotinylated secondantibody and the peroxidase-labeled avidin-biotin complex were incubatedon the sections for 30 minutes. The dilutions were made in PBS (pH 7.2),and all incubations were carried out in a moist chamber at roomtemperature. Between the different staining steps the slides were rinsedthree times with PBS. The peroxidase staining was visualised with a3-amino-9-ethylcarbazole (Sigma) solution (0.2 mg/ml in 0.05 M acetatebuffer containing 0.03% hydrogen peroxide, pH 5.0) at room temperaturefor 15 minutes. Finally, the sections were lightly counterstained withMayer's haematoxylin and mounted with aqueous mounting media (Aquamount,BDH). In control experiments the primary antibodies were replaced withthe IgG fraction of normal rabbit serum or the primary antibody waspreabsorbed with the RELP peptide. These stainings indicated thepresence of the RELP protein in a subset of duodenal epithelial cells,in a subset of stomach mucosal epithelial cells, in a subset of exocrinepancreatic ductal cells, in colon crypt bottom cells, in a subset ofmammary ductal epithelial cells, and in the epithelial cells of benignand malignant mucinous tumors originating from ovary, stomach, colon,breast, and pancreas, while the stroma remained completely negative. Theabundant and uniform expression of RELP protein in the epithelial cellsfrom mucinous tumors further supports the use of RELP as a tumor marker.As a secreted protein RELP can be measured from the serum or plasma.Moreover, anti-RELP antibodies might prove useful in detecting solitarytumor cells in tissue samples and cytologic specimen.

Example 6 Structure and Nucleotide Sequence of the Gene

RELP cDNA comprises 1517 nucleotides, and the protein coding region ismade up of 476 bp of nucleotides encoding a preprotein of 158 aminoacids. The 5′untranslated and 3′ untranslated regions contain 440 and601 nucleotides respectively. The first methionine (nt 441–443) ispreceded by a Kozaks' consensus translational start site. (Kozaksequence AAG before initiating methionine). A polyadenylation signal(AATAAA) is located 510 bp downstream of the termination codon. The genestructure of the protein was deduced by the analysis of genomicdatabases in the public domain. The missing base pairs flanking ends ofthe randomly ordered fragments of the genomic data base were acquired bysequencing these areas of the physical genomic RELP sequence.

A human genomic PAC clone containing the genomic RELP sequence wasobtained from GenomeSystemsInc (St. Louis, Mo.). NS3516 bacterial cellswere transformed with the PAC plasmid containing a genomic insert ofabout 120 kb. Plasmid DNA was isolated using EndoFree Plasmid Maxi Kit(Qiagen, Germany). The genomic sequence was amplified by PCR usingRELP-specific primers flanking the missing sequence data.

The primers used were as follows:

CAGCTGTGCTCCTGGATGGT Seq.ID No.12 TGGTCGGTACTTGCACAGGA Seq.ID No.13CTCCTATTGCTGAGCTGCCT Seq.ID No.14 ATTCGTTGCTGCTCCAAGTT Seq.ID No.15TTCCAGAAGCATGCGGCTG Seq.ID No.16 ACAGGAAGTGTTGGCGCTT Seq.ID No.17ATGGCTTCCAGAAGCATGC Seq.ID No.18 CTATGGTCGGTACTTGCACA Seq.ID No.19CTTGCTCTATGGTCGGTACT Seq.ID No.20 ACTGGGACCACTGGAGACACT Seq.ID No.21GAGACACTGAAGAAGGCAG Seq.ID No.22 AGACCCAGCTGTTTCATAGG Seq.ID No.23AATGGAGAGAGGGCAGAAGG Seq.ID No.24 TGATATCATCATGAGACCCAGCT Seq.ID No.25AGACAGTCATCCATTTGCCCA Seq.ID No.26 TGGGCAAATGGATGACTGTCT Seq.ID No.27CTCTAGAATCCAACAAAACTC Seq.ID No.28 TGCCAGACCAGGATCTGTACA Seq.ID No.29ATCCATATCGGCTGGCTTC Seq.ID No.30 CACTATGAAGAGAAGCCCCT Seq.ID No.31AAACACAACTGCTGCAGCGT Seq.ID No.32 GAAGCCAGCCGATATGGAT Seq.ID No.33TAGAGCTAGAAGCCACTACT Seq.ID No.34 TCCTGTGCAAGTACCGACCA Seq.ID No.35CAGTAGTGGCTTCTAGCTCT Seq.ID No.36 TCCTGGGCACTATGAAGAG Seq.ID No.37GGTAGCAATATTGTAGAATCC Seq.ID No.38 GTTTGTAGCACACTCCTGAT Seq.ID No.39TATGGCTGCAGTCTGCGGT Seq.ID No.40 ACTAGAGTGGTCATGGGAAC Seq.ID No.41GATTCCAGTTTGCAAGGTAC Seq.ID No.42 TACTGCTACTGCTGGGGAAT Seq.ID No.43

Amplified DNA fragments were subcloned into a TA vector and nucleotidesequences of the relp gene fragments were obtained by sequencing withvector-derived and relp specific primers. Comparison of genomic RELP DNAwith the RELP cDNA sequence revealed that the transcribed regions aredivided into seven exons separated by six introns and that allexon-intron junctions followed the GT-AG rule. The lengths of exons1,2,3,4,5,6,7 are of 172,174,161,98,137,106 and 669 bp respectively(FIG. 4). It was determined that due to differential splicing exon 2 isnot represented in all transcripts. The initiation of the first exon wasdeduced from the genomic sequence using the AG rule and the splice donoracceptor site consensus sequence location. Exons 1 to 3 encode the 5′untranslated region of 440 nt (or 266 nt in the splice variants wherethe exon 2 is missing) and exon 7 the 3′ untranslated region of 601 nt.

The promoter sequence of the relp gene was analyzed with the promoteranalyzing program Genomatix (http:genomatix.gsf.de/mat_fam). An Ap-1binding site and a cAMP responsive element are located at 15respectively 44 base pairs upstream from transcriptional initiationsite.

Example 7 Fluorescence in situ Hybridization (FISH)

To determine the chromosomal localization of the relp gene, fluorescentin situ hybridization (FISH) was performed. A human genomic PAC clonecontaining the RELP gene was used as a probe to localize RELP in humanchromosomes. The PAC plasmid was labeled with biotin-16-dUTP using nicktranslation. Slides with human interphase and metaphase nuclei werepretreated with 0.01 N HCl for 10 min at 37° C. and 0.01 N HClcontaining pepsin (20 mg/ml) for 5 min at 37° C. After dehydration ingraded ethanol, the slides were denaturated in 70% formamide/2×SSC at64° C. Hybridization was carried out at 37° C. overnight. Afterhybridization, the slides were washed in 2×SSC for 1×5 min at 45° C.,0.1×SSC for 2×5 min at 45° C. and in 4×SSC/0.2% Tween 1×5 min at roomtemperature, blocked in 5% BSA/4×SSC for 30 min at 37° C. and in4×SSC/0.2% Tween for 5 min at 45° C. Hybridized probes were detectedwith avidin-conjugated FITC and the signals were amplified withbiotinylated-anti-avidin antibodies. After washing at 45° C. in4×SSC/0.2% Tween for 3×5 min the slides were counterstained with DAPIand mounted in an antifade solution.

Hybridization showed exclusive signals on chromosome 1 band p12–13.1.

Example 8 Dot Blot and Northern Blot Analysis

Dot blot and Northern blot analyses were performed using Multiple TissueExpression (MTE) Array and Multiple Tissue Northern (MTN) blot II andIII (Clontech,). ³²P-labeled full length RELP cDNA was used as a probe.Labeling was done with the Multiprime DNA labeling system kit (AmershamPharmacia Biotech). For autoradiography filters were exposed to KodakBiomax MS film for 1–3 days. Dot blot analysis revealed RELP mRNA intissues of the gastrointestinal tract, in the prostate, and in testis.Northern blot analysis demonstrated high expression of a 1.5-kbtranscript in the duodenum, stomach, testis, and prostate. Significantexpression was also seen in the jejunum, ileum, ilocecum, appendix,descending colon and pancreas. No RELP expression was seen in thyroid,spinal cord, adrenal gland, bone marrow, spleen, thymus, ovary or bloodleukocytes.

The above is the description of the normal tissue distribution of RELP.

In the cancers identified in the body of the specification above, RELPis expressed ectopically, meaning that it is expressed in cells whichshould not express it at all, where its expression is irrelevant, and isdue to the regression of the level of differentiation. Thus, thepresence of RELP beyond normal levels is seen at the level of the wholeorganism: the body produces too much RELP (measured in plasma), whichindicates that there is a cancer in one of the organs known to developRELP-positive tumors.

Example 9 Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Reverse transcription and PCR amplification of RELP mRNA was performedby continous RT-PCR using the Robust RT-PCR kit (Finnzymes, Espoo,Finland). One hundred ng of poly(A) RNA was reverse-transcribed intocDNA for one RT-PCR reaction. The primers used were as follows:

sense: CAGCTGTGCTCCTGGATGGT, Seq.ID No.12 CTCCTATTGCTGAGCTGCCT Seq.IDNo.14 antisense: TGGTCGGTACTTGCACAGGA, Seq.ID No.44 ATTCGTTGCTGCTCCAAGTTSeq.ID No.45

Reverse transcription reaction was performed at 48° C. for 30 min.Before PCR amplification, the samples were initially denatured at 95° C.for 4 min. Cycling parameters were as follows (30×): denaturation at 95°C. for 30 s, annealing at 60° C. for 1 min, elongation at 72° C. for 1min and final extension at 72° C. for 5 min.

Amplified products were analyzed by agarose electrophoresis andsubcloned according to manufacturer's instructions into a vector of theTA cloning system (Invitrogen, San Diego). Nucleotide sequencing of thecloned PCR products were performed by the Thermo Sequenace Kit(Amersham, Buckingshire, UK) and an ALF express sequenator (Pharmacia,Uppsala, Sweden). The procedure verified the transcription of RELP induodenum, colon, stomach, and pancreas, and excluded the possibilitythat the Northern blot and Dot blot experiments should have detected RNArepresenting other reg proteins that are homologus to RELP.

Example 10 In vitro Translation

A cDNA fragment containing the full length sequence of RELP cDNA wassubcloned into the eukaryotic expression vector pcDNA 3 (Invitrogen, SanDiego) under the T7 RNA polymerase promoter. The RELP protein wasexpressed using Rabbit Reticulocyte Lysate with Canine PancreaticMicrosomal Membranes (Promega, Madison, Wis.) in the presence of³⁵S-methionine (Amersham International's Redivue L-35Smethionine,Amersham Pharmacia Biotech). Proteins obtained by in vitro translationwere analyzed by SDS-PAGE (12%) gel electrophoresis and visualized byautoradiography. The translation resulted in a protein product with anapparent molecular weight of 18 kd as analysed by PAGE. This is inconcordance with the calculated molecular weight of RELP (18.2 kd). Whenthe microsomal membrane fraction was added, the size of the proteinproduct was reduced to 17 kd, which is in concordance with the predictedstructure of RELP, including an N-terminal cleavable 23 amino acidsignal peptide.

Example 11 Enzyme Immunoassay (Prophetic)

Immunoassays are prepared for the RELP antigen. This is achievable sincedetection of 10 fmol/L is possible in competitive assays. Sensitivity ofnoncompetitive assay is determined by lower limit of detection of thelabel used: 1 to 2,000,000 Zeptomoles (10⁻²¹ moles). “Tietz Fundamentalsof Clinical Chemistry” 4th Edition, p143

To develop an Enzyme Immunoassay (EIA) procedure, antigen standardscomprising a digest of colon tumor specimens (shown to contain theantigen by immunoperoxidase staining) are used. Human primary coloncancer specimens are pooled and homogenized in 10 volumes of 10 mM Trisbuffer, pH 7.4, containing 0.2% (w/v) sodium deoxycholate at 4C. Thehomogenate is quickly brought to 37 C and the following reagents (finalconcentration) are added while stirring: 1 mM cysteine (Sigma), 1 mMEDTA (Sigma), and papain (0.8 unit/ml) (Boehringer-Mannheim,Indianapolis, Ind.). After 5 minutes, digestion is stopped by theaddition of 5 mM iodoacetamide (Sigma). The homogenate is centrifuged at100,000×g for 1 hour at 4C, then extensively dialyzed against 10 mMTris/0.9% NaCl solution buffer, pH 7.4, containing phenylmethysulfonylfluoride and aminocaproic acid, each at 10 mM. The homogenate is frozenin small aliquots at a concentration of 0.5 mg of protein/ml.

The dose response curve that will be generated for the immunoassayprocedure measuring RELP demonstrates linearity between antigen input of100 ng to 100 μg/ml. For serum analysis, the range is 1 ng to 1000ng/ml, since these samples are diluted 10-fold prior to assay.

Solid-phase preparations of the antibodies described in Example 2 areprepared using CNBr-activated Sepharose (Pharmacia). Microtiter plates(Nunc I Immunoplates; Grand Island Biological Co., Grand Island, N.Y.)are coated with the antibodies (200 μl/well) in 50 mMcarbonate-bicarbonate buffer, pH 9.6, for 18 hours at 4C. After removalof the antibody solution, residual protein binding sites on the plasticare blocked by the addition of 200 μl of assay buffer [PBS containing 1%(v/v) rabbit serum and 1% (w/v) bovine albumin]. After 1 hour ofincubation at room temperature, the coated plates are used immediatelyfor the assay procedure.

To perform the assay, 200 μl samples, diluted in assay buffer, areapplied for 1–5 hours at 37C. After 3 washes using assay buffer, 200 μlof the antibody covalently conjugated to horseradish peroxidase (Sigma,Type VI) is applied to each well for 1.5 hours at 37C. The conjugate isdiluted to a concentration of 0.5 μg of immunoglobulin per ml of PBScontaining 10% (v/v) murine serum. Following a wash procedure as above,200 μl of substrate per well are applied for 0.5 hours at roomtemperature. Substrate solution contains 0.4 mg of o-phenylenediamineper ml of pH 5.0 citrate buffer and 0.003% hydrogen peroxide. Thereaction is stopped by addition of 50 μl of 2N sulfuric acid, andabsorbance is monitored at 488 nM using an enzyme assay plate reader(Fisher Scientific Co., Pittsburgh, Pa.).

The percentage of bound enzyme conjugate is calculated by the formula:(B−B₀)(B_(t)−B₀)(100)where B=absorbance of the sample, B_(t)=maximal absorbance, andB₀=absorbance of the blank. Each assay is performed in triplicate usinga standard digest and 26-fold diluted serum samples diluted in assaybuffer. Specificity of the immunoassay is examined by substitutingvarious antibody reagents at the solid phase, including an antibody toCEA and nonimmune rabbit serum. Of the solid phase antibodies onlyantibody prepared according to Example 2 binds antigen at highdilutions.

Levels of serum RELP are detected for normal control subjects, patientswith benign and malignant prostate diseases and patients with ovarian,stomach, colon, and breast cancer.

Sera obtained from apparently healthy individuals exhibits a mean valueof approximately 90 ng/ml of RELP/ml. Only 5% of the samples expressserum antigen at 150 ng/ml or above, and this value is chosen as thecutoff for elevated serum levels.

Sera from patients with benign disease of the colon exhibit a mean RELPvalue of 160 ng/ml. The incidence of values above 200 ng/ml is 5%.Patients with colon cancer (with evidence of disease) exhibit a widerange of circulating levels of RELP with a mean value above 160 ng/ml.

Sera obtained from patients with cancers corresponding to thosedescribed above are also evaluated. The incidence of elevated RELPvalues is 90%. Mean serum values from the group with cancer aresignificantly higher than control levels (about 250% higher).

Using a limited number of postoperative colon cancer patients withprimary localized disease, a significant decrease in serum RELP occurs.These data indicate a relationship between serum RELP levels and tumorload. Such measurements are thus valuable for patient monitoring.

1. A method for detecting the presence of ovary, breast or prostatecancer, comprising detecting the concentration of the RELP of SEQ IDNO:2 in an isolated serum sample, wherein the concentration of the RELPof SEQ ID NO:2 in said sample above the normal controls correlates withthe presence of said cancer.